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Description of key information

SFL is not considered to be irritating to the skin or eyes.  

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 October 2010 - 18 October 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
other: OECD Guidelines No. 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: Reconstituted skin membranes
Strain:
other: Reconstituted skin membranes
Details on test animals and environmental conditions:
The EpiDerm™ EPI-200 skin model consists of normal human epidermal keratinocytes (NHEK) from one single donor, derived from neonatal-foreskin tissue. The keratinocytes are plated on chemically modified, collagen-coated, 8 mm ID cell culture inserts (surface area 0.5 cm2). The skin models are commercially available and were obtained from MatTek Corporation, USA. Upon arrival at TNO Quality of Life on 28 April, the EPI-200 skin models were pre-incubated in 0.9 mL assay medium for 60 min (at ca. 37 ºC and ca. 5% CO2). At the end of the first pre-incubation period, the tissues were additionally pre-incubated overnight for 19±1 h (at ca. 37 ºC and ca. 5% CO2).
Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
other: phosphate buffered saline
Controls:
other: Positive control: 5% Sodium Lauryl Sulphate (SLS) in demineralised water (=5% Sodium Dodecyl Sulphate (SDS)); Negative control: Phosphate Buffered Saline (PBS)
Amount / concentration applied:
The test substance has a clay-like appearance. Therefore, just before test substance application, 25±1 mg of the test substance was mixed with 25 μL phosphate buffered saline, to facilitate dosing and equal distribution of the test substance. During the main study it was not possible to transfer the complete mixture to the skin surface and some test substance remained in the vial. Consequently the total amount that was applied to the skin membrane was less than 25 mg. Since the in vitro skin irritation test is based on the application of an excess amount of the test substance, and since the skin surface was completely covered with Sugar Factory Lime in the present study, it was concluded that the skin membranes were sufficiently exposed to the test substance.
Duration of treatment / exposure:
The test substance was applied topically to the skin membranes for 60 minutes and then incubated for a 49 hour period.
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
STUDY DESIGN
After overnight pre-incubation of the tissues, as much as possible of the test substance / phosphate buffered saline mixture was transferred to the skin membranes or skin membranes were exposed to 30 μl of the positive or negative controls. Skin membranes were exposed to the study substances in triplicate for 60±1 min. Frozen controls were exposed to the test substance and negative control in duplicate.

Exposure was performed at ambient temperature in the flow cabin. After dosing the last tissue, the tissues were transferred to a humidified incubator (ca. 37 ºC and ca. 5% CO2). After 35±1 min, the tissues were removed from the incubator and placed in the flow cabin until the exposure period of 60 min was completed for the first dosed tissue. When the exposure period was completed, the inserts were removed from the well and the skin surface was carefully washed using excess of phosphate buffered saline (PBS). Subsequently, the insert was blotted dry and the tissue surface was carefully dried using a sterile cotton swab. The insert was then transferred to clean 6-well plate containing fresh medium (0.9 mL/well). Medium was refreshed after 22h. Following an additional 27h incubation period at ca. 37 °C and ca. 5% CO2 in a humidified incubator, viability was determined using the MTT assay.

MTT ASSAY
An MTT solution of 1 mg/mL was prepared by diluting MTT concentrate 5 times in MTT diluent. The bottom of the inserts was blotted dry and the inserts placed in a 24-well plate containing 300 μL of MTT medium/well. The plates were placed in an incubator at ca. 37 ºC and ca. 5% CO2. After 180±1 min, the tissues were rinsed three times with PBS. The formazan product was extracted from the tissue using 2 mL MTT extractant. Extraction was performed in the dark for 2 days at 2-10 ºC.
After completion of the extraction period, the skin membrane was pierced with a needle to allow the extract to run into the well from which the membrane was taken. The optical density was measured in triplicate in 200 μL sub-fractions using a spectrophotometer set at 570 nm. Extractant solvent alone was used as blank. The mean optical density was calculated and expressed as the percentage viability compared to the negative control.
Irritation / corrosion parameter:
other: other: Viability of cells
Value:
73
Remarks on result:
other:
Remarks:
Basis: other: Mean tissue viability (%). Time point: Not applicable. Reversibility: other: Not applicable. Remarks: Results for SFL (% of negative control). (migrated information)
Irritation / corrosion parameter:
other: other: Viability of cells
Value:
100
Remarks on result:
other:
Remarks:
Basis: other: Mean tissue viability (%). Time point: Not applicable. Reversibility: other: Not applicable. Remarks: Results for negative control. (migrated information)
Irritation / corrosion parameter:
other: other: Viability of cells
Value:
9
Remarks on result:
other:
Remarks:
Basis: other: Mean tissue viability (%). Time point: Not applicable. Reversibility: other: Not applicable. Remarks: Results for positive control, 5% SLS (% of negative control). (migrated information)
Irritant / corrosive response data:
Optical density of the negative controls (PBS) and positive controls (5% SLS) were within the acceptable range and correctly indicated non irritancy and irritancy, respectively. Optical density of the extractant solvent alone was < 0.1 (0.040). This demonstrated validity of the study.

Frozen controls exposed to the test substance showed a slightly higher MTT conversion than the frozen controls treated with the negative control. True viability of tissues treated with the test substance was corrected for the MTT conversion by the test substance.

Based on the results, the test substance Sugar Factory Lime (SFL) was classified as non-irritant.

Table 3: Formazan production in EpiDermskin membranes exposed to the test substances, negative and positive controls (results of MTT assay)

Test group

Study substance

A570 (% of control)

NC

Negative control (PBS)

100

A

Sugar Factory Lime (SFL)

73

PC

Positive control (5 % SLS)

9

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Sugar factory lime (SFL) is not classified as a skin irritant.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 February-28 February, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK.
- Age at study initiation: 12-20 weeks
- Weight at study initiation: 2.12 and 2.58 kg
- Housing: individually housed in suspended cages
- Diet: 2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK
- Water: ad libitum mains drinking water
- Acclimation period: 5 days minimum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23
- Humidity (%): 30-70
- Air changes (per hr): 15/h minimum
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 11/02/2013 - 28/02/2013
Vehicle:
unchanged (no vehicle)
Controls:
other: left eye of each animal used as control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.1 mL (97 mg)
Duration of treatment / exposure:
Animals were exposed to the test material for up to 72 hours.
Observation period (in vivo):
1, 24, 48 and 72 hours following treatment
Number of animals or in vitro replicates:
2
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Not applicable
- Time after start of exposure: Not applicable

SCORING SYSTEM: Draize

TOOL USED TO ASSESS SCORE: Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
other: Mean of 24, 48 & 72 hrs
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
other: Mean of 24, 48 & 72 hrs
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #1
Time point:
other: mean of 24, 48 & 72 hrs
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #2
Time point:
other: Mean of 24, 48 & 72 hrs
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
other: Mean of 24, 48 & 72 hrs
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 72 hrs
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
other: Mean of 24, 48 &72 hrs
Score:
0.3
Max. score:
3
Reversibility:
fully reversible within: 48 hrs
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: Mean of 24, 48 & 72 grs
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 48 hrs
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: Mean of 24, 48 & 72 hrs
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 48 hrs
Other effects:
Both animals showed expected gain in body weight during the study.

Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex

72960 Male

73002 Male

IPR = 1

IPR = 1

Time after treatment

1 h

24 h

48 h

72 h

1 h

24 h

48 h

72 h

CORNEA

0

0

0

0

0

0

0

0

E = Degree of Opacity

0

0

0

0

0

0

0

0

F = Area of Cornea Involved

0

0

0

0

0

0

0

0

Score (E x F) x 5

0

0

0

0

0

0

0

0

IRIS

 

 

 

 

 

 

 

 

D

1

0

0

0

0

0

0

0

Score (D x 5)

5

0

0

0

0

0

0

0

CONJUNCTIVAE

 

 

 

 

 

 

 

 

A = Redness

2

2

1

0

2

1

0

0

B = Chemosis

2

1

0

0

1

1

0

0

C = Discharge

1

0

0

0

1

0

0

0

Score (A + B + C) x 2

10

6

2

0

8

4

0

0

Total Score

1

6

2

0

8

4

0

0

IPR = Initial pain reaction; Class 1 – Reaction by animal = A few blinks only, normal within one or two Minutes; Descriptive reading = Practically no initial pain

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
SFL is not irritating to the eyes of rabbits.
Executive summary:

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit. A single application of the test item to the non-irrigated eye of two rabbits produced iridial inflammation and moderate conjunctival irritation. One treated eye appeared normal at the 48-Hour observation and the other treated eye appeared normal at the 72-Hour observation. The test item produced a maximum group mean score of 11.5 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system. The test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008, relating to the Classification, Labeling and Packaging of Substances and Mixtures.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

An in vitro skin irritation study was performed with Sugar Factory Lime (SFL) using reconstituted skin membranes in accordance with OECD Guideline 439 and GLP [Reus (2010)]. The test substance was applied topically to the skin membranes for 60 minutes. After a 49 hour incubation period, the effect on the tissue viability was determined, based on the reduction of MTT to a purple formazan precipitate by mitochondrial dehydrogenase activity. The relative mean viability of the test material treated tissues was 73%. Hence, SFL is considered to be non-irritating to skin.

This study is supported by a reliable GLP OECD guideline 404 in vivo skin irritation study [Sanders (2004a)] where 0.5 g precipitated calcium carbonate (PCC) was applied to the shaved skin of 3 New Zealand White rabbits under a semi-occlusive patch for 4 hours. Animals were observed after removal of the patch at 1, 24, 48 and 72 h. The average of the erythema and oedema assessments for the 3 animals after 72 h was 0.0. Therefore, calcium carbonate was found to be non-irritating to the skin in rabbits.

 Eye irritation:

In a reliable in vivo eye irritation study performed according to OECD TG 405 under GLP 0.1 mL (97 mg) of Sugar Factory Lime (SFL) was instilled into the right eye of two New Zealand White rabbits [Harlan Laboratories (2013e)]. Ocular observations were made at 1, 24, 48 and 72 h after instillation. No corneal effects were noted during the study. Iridial inflammation was noted in one treated eye one hour after treatment. Moderate conjunctival irritation was noted in both treated eyes one hour after treatment. Minimal conjunctival irritation was noted in both treated eyes at the 24-hour observation and in one treated eye at the 48-hour observation. The mean scores for 24, 48 and 72 hours for each animal were below the limits for classification as irritating to eyes (Category 2) and the effects were fully reversible within 72 hours. Based on the results of this study, SFL is not irritating to rabbit eyes.

The results of this study are supported by an in vitro study performed using SFL and an in vivo study performed using calcium carbonate, the major constituent of SFL, described below.

An in vitro eye irritation study was performed with SFL using the Chicken Eye (ICE) test, in accordance with GLP [Prinsen (2010)]. The neat test substance (30 mg) was applied in one single dose onto the cornea of isolated eyes. SFL caused very slight swelling, slight opacity and slight or slight to moderate fluorescein retention. The calculated Irritation Index was 51. After the 30-second application SFL caused comparable signs of irritation. Based on the results of this in vitro study, SFL is not irritating to eyes.

In a reliable study performed according to OECD TG 405 under GLP [Sanders (2004b)] approximately 100 mg precipitated calcium carbonate (PCC) was instilled into the eyes of 3 New Zealand White rabbits. Ocular observations were made at 1, 24, 48 and 72 h after instillation. There were no corneal or iridial effects or chemosis noted during the study after 24 hours of exposure; hence, the mean scores for 24, 48 and 72 hours for each animal were 0. The mean scores for 24, 48 and 72 hours for each animal for conjunctivae redness, although above 0, were below the limits for classification as irritating to eyes (Category 2) and the effects were fully reversible within an observation period of 21 days. Based on the results of this study, calcium carbonate is not irritating to rabbit eyes.


Justification for selection of skin irritation / corrosion endpoint:
Study performed on SFL

Justification for selection of eye irritation endpoint:
In vivo study performed on SFL

Justification for classification or non-classification

SFL is not classified as a skin irritant based on the results of an in vitro study on SFL itself. This is supported by a negative result in an in vivo skin irritation study performed using calcium carbonate, the major constituent of SFL.

SFL is not classified as an eye irritant based on the results of an in vivo eye irritation study.